Peracetic acid composition

ABSTRACT

The invention relates to anti-microbial compositons useful against a wide range of microoganisms undesirable on a wide variety of materials, including food, food contact and non-food contact surfaces, and surfaces in industrial, recreational, health care, and other institutional environments. More particularly, the anit-microbial compositions comprise peracetic acid in combination with a)citric acid or a salt and b) salicyclic acid or a slat in aqueous solution.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/876,941, filed Dec. 22, 2006.

FIELD OF THE INVENTION

The invention relates to anti-microbial compositions useful against awide range of microorganisms that are undesirable on a wide variety ofmaterials, including food, food contact and non-food contact surfaces,and surfaces in industrial, recreational, health care, and otherinstitutional environments. More particularly, the anti-microbialcompositions comprise an aqueous solution of peracetic acid (PAA) and anadjuvant, the adjuvant comprising a) citric acid or a salt of citricacid and b) salicylic acid or a salt of salicylic acid.

BACKGROUND OF THE INVENTION

Certain environments pose challenges to the efficacy of sanitizers anddisinfectants upon various microorganisms found within thoseenvironments. Additionally, a number of widely applied antimicrobialcontrol agents have effectively been reduced because of their inherenttoxicity, long term and latent effects on the environment, food chain,and surface waters. A number of compounds are undergoing increasedscrutiny because of these factors coupled with misapplication andgeneral in-use safety.

Peracetic acid is known to be a useful antimicrobial agent. However, itsantimicrobial effect is best obtained with high concentrations(generally greater than 100 parts per million (ppm)). At theseconcentrations the material has an overbearing odor, sometimes causesoxidative damage to foodstuffs and surfaces to which it is applied, andpresent hazards to persons handling the materials. US 4,051,058discloses aqueous solutions of peracetic acid in concentrations of 0.5%to 20% by weight for use in sanitizing and disinfecting applications.Compositions additionally containing organic acids are also known. U.S.Pat. No. 6,617,290 discloses the use of acidifying agents classified asGRAS (Generally Regarded As Safe) for use as food additives inpreparations for cleaning and sanitizing food contact and non-foodcontact surfaces. These agents include citric and lactic acids. US6,475,967 discloses a light duty antibacterial liquid detergentdisplaying foaming and grease-cutting as well as low corrosiveproperties. The aqueous composition comprises hydroxyl-containingorganic acid(s), a peracetic acid, and other components includingvarious surfactants, and polyethylene glycol.

A need continues to exist for antimicrobial compositions that arestable, environmentally compatible, are “no rinse” after application,exhibit residual antimicrobial activity, do not alter organolepticproperties such as taste, smell, or visual appearance of food whichcomes into contact with the composition, will not oxidize hard surfaces,and will not adversely affect humans should incidental ingestion orcontact occur. Particularly desirable are antimicrobial compositionsthat are effective at low concentrations in solution.

SUMMARY OF THE INVENTION

The invention is a cleaning and sanitizing composition comprising, inaqueous solution,

-   -   a. peracetic acid; and    -   b. an adjuvant comprising (i) salicylic acid or its salts        and (ii) citric acid or its salts.

The invention further includes the adjuvant wherein the salicylic acidand citric acid or their respective salts are dissolved to theirsolubility limits and in proportionate ratios ranging from about 1:5 to1:1 by weight respectively. One or more stabilizing agents, wettingagents, hydrotopes, thickeners, foaming agents, acidifiers, pigments,dyes, surfactants, or combinations thereof may further be components ofthe invention to effect other properties desirable in such acomposition. One such surfactant consists of one or more anionic ornonionic surfactants in concentrations up to 2% selected from the groupFMC HRSTM, polyoxylene (C₂₀ to C₈₀) sorbitan monooleates, alklyl longchain fatty acid (C₅ to C₂₀) metallic esters and alcohols; andalkylbenzenesulfonic acids and alcohols and/or their metallic estersalts having alkyl groups ranging from C₂ to C₂₀.

The invention further includes a kit comprising

-   -   a. an aqueous peracetic acid solution; and    -   b. a mixture comprising salicylic acid and citric acid or their        respective salts in proportionate ratios ranging from about 1:5        to 1:1 by weight respectively and in a solution matrix acting as        solvent of glacial acetic acid (70-90%) and water (30-5%).

The invention also encompasses a method of reducing microbialcontamination comprising applying the anti-microbial composition to asurface in an amount and for a time sufficient to reduce the microbialcontamination. The surface to which the composition is applied is any ofmeat, poultry, seafood, or portions thereof. The surface mayalternatively be a plant material (leaf, stalk, flower, fruit,vegetable, or portion thereof). Additionally, the surface may be anon-food surface. The method further contemplates recovering the appliedcomposition and reprocessing the recovered composition to yield arecycled anti-microbial composition. The method includes an embodimentwherein the peracetic acid component is present at end useconcentrations ranging up to 1%. The invention further contemplates acomposition wherein the sodium salicylate or salicylic acid is presentat end use at up to 20000 ppm. The invention further contemplates acomposition wherein the citric acid is present at end use at up to 20000ppm. End use concentrations of the composition generally have pH valuesbetween 1 and 4.5.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to improved mycocidal and bacteriocidalcompositions that are safe for food contact and non-food surfaceswithout requiring a post-sanitizing rinse and are safe for incidentalhuman contact. The composition of the invention is intended to enhancethe efficacy of peracetic acid solutions at end use concentrations. Theinvention is a composition comprising:

-   -   a) an aqueous peracetic acid solution; and    -   b) an adjuvant comprising (i) salicylic acid or its salts        (cationic valence +1 or +2) and (ii) citric acid or its salts        (cationic valence +1 or +2).

The aqueous adjuvant solution comprises 70-95% acetic acid and 30-5% byweight water containing the salicylic acid and the citric acidcomponents dissolved in the liquid. The resulting adjuvant is then addedto the peracetic acid/water solution such that the upper ppm limit ofPAA is about 10,000 ppm and the upper ppm limit of the other acids isabout 40,000 ppm. One method of forming the invention includes adding tothe peracetic acid formulation in situ salicylic acid or its salts (as asimple form of an aromatic carboxylic acid) and citric acid or its salts(as a tricarboxylic acid). Salicylic acid and citric acid are used toform the adjuvant in concentrations of from about 100 ppm to about40,000 ppm. Above this concentration the additive formulation itselfbecomes unstable (acetic acid+water+sodium salicylate+citric acid) tosupport addition into the PAA/water solution.

The resulting peracetic acid in situ mixture containing the adjuvantprovides enhanced performance and latent residual activity on a treatedsurface once the liquid portions have disappeared through decompositionand evaporation. Additional components may be anionic or non-ionicsurfactants which can foam and thereby hold the sanitizer in place forextended periods of time. The composition contains peracetic acid at enduse concentrations of between about 40 ppm to about 10,000 ppm. Anylower use concentrations of peracetic acid require too long of a contacttime to be considered practicably effective. The final diluted solutionas applied to a food stuff or surface will contain by weight in oneembodiment approximately water 84.5 -91.4%; acetic acid 10.1-0.149%; PAA1-0.004%; hydrogen peroxide 4-0.003%; citric acid or salt 0.56-0.010%;and, salicylic acid or salycilate salts 0.12-0.002%. All percents hereand elsewhere throughout the application are percents by weight unlessindicated otherwise.

The compositions of the invention are appropriate for use in food andbeverage processing plants (including facilities related to meat,seafood, poultry, produce, dairies, wineries, breweries, and beverageplants), food preparation kitchens, food serving establishments,nursing-care and hospital-care applications, and industrial andinstitutional surfaces where concerns exist regarding the safety of foodproducts, environmental residues, or for human contact. The compositioncan be applied either through in situ mixing via batch tank, in-linemixing with ratio controllers or ratio control via pump speeds, ortopical application of one liquid separately into/onto one another uponthe surface to be treated. The invention is also provided in the form ofa kit containing an aqueous peracetic acid solution as a first componentand a concentrate of salicylic and citric acid or salts thereof as asecond component.

Although not wishing to be bound to any particular theory, the followinginformation is presented to assist those skilled in the art tounderstand the applicability and limits of this invention.

Yeasts and molds have a typical surface structure containing a cell wallgenerally comprised of 1,3-{acute over (α)}-peptidoglycans and/or chitinalong with the lipid bi-layer associated with cell membranes. Theslightly different construction of fungal cell walls relative to the 1,4and 1,6-{acute over (α)}-peptidoglycans associated with bacteria havingcell walls (gram positive) presents some different characteristics whenattempting to destroy the organism.

The intent of the invention is to disrupt the metabolic homeostaticmechanisms of the target organisms with a mixture of antimicrobialcompounds. All antimicrobial agents will have some toxic effects tolower and higher forms of life, including animals and humans, becauseall life forms share some degree of common metabolic pathways. Typicalhomeostatic metabolic mechanisms include: protein coagulation, lysing,oxidation, reduction, and competitive and non-competitive inhibition.More specifically, citric acid disrupts respiratory pathways via Kreb'scycle and has known preservative properties. Salicylic acid derivativesinhibit folic acid synthesis in mycobacterium and are bacteriostatic.These derivatives are known for their use as preservatives, fungicides(i.e., as a treatment of tinea), and have mild antibiotic propertieswhen used as a topical treatment for skin ailments. These compoundsweaken the target organism and so enhance the efficacy of peracetic acidin oxidative lysing.

Cell damage is distinguished as “-static” or “-cidal” activity based onthe degree of the control agent's efficacy and defined by officiallaboratory control protocols. “Static” cell damage is reversible, withthe organism able to multiply once relieved of exposure to the agent,“-cidal” cell damage is irreversible, with lethality resulting fromcomplete microbial cell destruction or incapacitation. According to thisdefinition, preservatives are generally considered inhibitory,bacteriostatic, or mycobacteriostatic and sanitizers and disinfectantsare considered microbiocidal.

Preservatives are inhibitory compounds added to foods and non-foodproducts to prolong or enhance shelf-life by hindering the deteriorationof properties such as: texture, nutritive value, odor, flavor, color,appearance, value or safety. Minimum Inhibitory Concentration is thestandard criteria for evaluating such materials as is Zone ofInhibition. The key differentiators between preservatives and sanitizersare: 1) mode of action—preservatives prevent growth instead ofdemonstrating lethality; and 2) exposure time—preservatives operate overperiods of days to months whereas sanitizers must provide 99.9999% (5log order) lethality within 30 seconds at nominal 20 degrees C.

Sanitizers reduce microbial contaminants to levels deemed safe as givenby public health requirements and must result in 99.9999% reduction (5log order reduction) from a practical perspective for given organisms asdefined by Germicidal and Detergent Sanitizing Action of Disinfectants,Official Methods of Analysis of the Association of Official analyticalChemists, paragraph 960.09 and applicable sections, 15^(th) Edition,1990 (EPA Guideline 91-2). Disinfectants must pass a more stringentantimicrobial test; the A.O.A.C. Use Dilution Methods, Official Methodsof Analysis of the Association of Official analytical Chemists,paragraph 955.14 and applicable sections, 15^(th) Edition, 1990 (EPAGuideline 91-2).

It is desirable that sanitizing agents or compounds have key propertiesbeyond antimicrobial efficacy. They include “no rinse” after applicationand exhibit residual activity where “residual activity” refers to somelatent antimicrobial effects remain on the treated surface ifcontaminated by microorganisms during storage or lag periods by means ofa lingering coating or film.

Additionally, the sanitizer ought not to alter food or (organolepticproperties such as taste, smell, or visual appearance) if food contactoccurs and should not affect humans should incidental ingestion result.

Compositions:

The compositions of the present invention comprise (a) peracetic acid(b) salicylic acid or a salt thereof and (c) citric acid or a saltthereof.

Peracetic acid is well known for its highly oxidative properties and asa bactericide and fungicide, especially in food processing operations.VigorOx® Liquid Sanitizer & Disinfectant and VigorOx® LS (LS&D and LS,FMC, USA) are equilibrium mixtures of peracetic acid, hydrogen peroxide,acetic acid, water and stabilizer. They are used at low solutionconcentrations to rapidly kill a broad spectrum of microorganismsincluding gram-positive and gram negative bacteria. They are moretolerant than most sanitizers to pH, temperature, water hardness, andorganic soils. The products are EPA-registered solutions for the foodand beverage industry. VigorOx® LS&D′s and VigorOx® LS's no-rinse,non-foaming formulas and their ability to sanitize at cold temperaturesmake them ideally suited for use in circulation clean-in-place (CIP)systems for food processing equipment. VigorOx® LS&D and VigorOx® LS arealso approved for immersion cleaning of equipment and utensils in foodprocessing and packaging plants and eating establishments. VigorOx® LS&Dis composed of: PAA 5-6%; H₂O₂ 21-23%; acetic acid 10-11%; and water63-65%. Clarity®, Blitz®, and Spectrum® are additional FMC formulationsof peracetic acid. The compositions for Clarity®, Blitz®, Spectrum®, andVigorOx® SP-15 are identical with registration differences for theirintended use: PAA 15-17%; H₂O₂ 9-11%; acetic acid 33-38%; sulfuric acid0-1%; and water 30-44%.

Salicylic acid is well known as the substructure for common aspirin, iseasily metabolized at low exposure rates, and is known as a mildantibiotic for skin-care products targeting acne, psoriasis, calluses,corns, keratosis pilaris, and warts. It is also known for inhibitinggenes necessary for pathenogensis in some soil microbes, such asPseudomonas aeruginosa.

Citric acid and its salts are well established as preservatives in food,especially beverages, and as pH buffers in solutions. Citric acid isGenerally Regarded As Safe (GRAS) by the FDA.

In addition to the free citric or salicylic acids, the compositions ofthe present invention may alternatively contain “salts” of these acids.The salts may be salicylate salt(s) comprising cationic salts including,but not limited to, sodium, potassium, ammonium; salicylates salts ofcations with valences of +1 and +2, or alkyl (C₂ to C₂₀), or acetylderivatives and/or with side chains in the ortho, meta, and/or parapositions of alkyl, aryl, acetyl, or carboxy side chains of length C₂ toC₂₀. Salts may also be citric acid ester salts of cations with valencesof +1 and +2 including complex cations such as ammonium. A preferredcomposition comprises peracetic acid, sodium salicylate and citric acid.

The compositions of the present invention upon drying will leave minuteamounts of salicylic acid or its salts and citric acid or its saltsafter the peracetic acid has decomposed or has been lost by evaporation.This effect will provide for some extended latent antimicrobial effects.

Additives:

The compositions may further comprise a variety of other optionalingredients such as surfactants, wetting agents, hydrotopes, thickeners,foaming agents, acidifiers, builders, stabilizers, bleach activators,soil suspenders, dye transfer agents, brighteners, perfumes,anti-dusting agents, enzymes, dispersants, dye transfer inhibitors,pigments, odor masking agents, or a combination thereof.

Surfactants of particular interest are anionic and nonionic surfactantsused in concentrations from 0 to 2%, such as, but not limited to, FMCHRSTM, polyoxylene (C₂₀ to C₈₀) sorbitan monooleates, alklyl long chainfatty acid (C₅ to C₂₀) metallic esters and alcohols; andalkylbenzenesulfonic acids and alcohols and/or their metallic estersalts having alkyl groups ranging from C₂ to C₂₀.

The invention is preferably formulated as a liquid composition,preferably an aqueous composition. The pH of the composition ispreferably between 2-5 pH and may be maintained by adjustment with PAA,citric, or acetic acid or mixture of these acids. The PAA and adjuvantsolution can be combined into one solution to which can be added afoaming agent, (i.e., FMC HRSTM) at a rate of 2-10 liquid ounces pergallon of sanitizer solution and applied by means of devices andequipment known in the art. The invention may also be combined by meansof a two-part spray system (PAA solution and adjuvant) to surfaces. PAAformulations are generally marketed as concentrates. The adjuvant of theinvention can therefore be provided as an additive formulation. Anillustrative formulation is a 10× concentration consisting ofapproximately 79% acetic acid, 14.2% water, 5.6% citric acid, and 1.2%Na salicylate. Additionally, the adjuvant of the invention may beprovided as a dry powder having a ratio of citric acid to Na salicylateof 4.67:1 by weight.

Target Organisms:

The compositions of the invention arc effectively used to sanitize ordisinfect food and surfaces where microbial populations are the resultof contamination by fecal matter or digestive tract content.Additionally, contamination by other microbial vectors is alsoeffectively reduced or eliminated by applying the disclosedcompositions. The invention is effective in combating a variety ofmicroorganisms including, but not limited to:

Gram positive Bacillus, Listeria, Staphylococcus, Streptococcus,bacteria Enterococcus, and Clostridium. Gram negative Escherichia,Salmonella, Pseudomonas, Moraxella, bacteria Heliobactor,Stenotrophomonas, Acetobactoer, and Bdellovibrio. Viruses Avian viraltypes and influenza species Fungi & Molds Penicillium spp., Geotrichumspp., Aspergillus spp. Yeasts Candida spp., Rhyzopus spp., Mucor.

The microorganisms used in developing this invention and in the examplesbelow were obtained from the American Type Culture Collection (ATCC),Manassas, Va. 20108 or from Remel Microbiology Products, Lenexa, Kans.,66215 USA. Specific organisms studied with use of the AOAC Use-DilutionMethod in the development of this invention were Pseudomonas aeruginosa(ATCC 15442), Staphylococcus aureus (ATCC 6538), Salmonella choleraesuis(ATCC 10708). Specific organisms studied for germicidal and detergentsanitizing action were Escherichia coli (ATCC 11229) and Staphylococcusaureus (ATCC 6538). Additionally studied in a moist environment incontact with fruit juice were species C. parapsilosis and M. plumbeus.

Types of Environments and Methods of Application: The compositions ofthis invention are suitable for use in a wide variety of environmentsincluding circulation cleaning in place (CIP) and industrial sanitizingof equipment such as tanks pipelines, evaporators, fillers,pasteurizers, aseptic equipment, and for sanitizing previously cleaned,hard, porous and non-porous food contact surfaces of equipment in, forexample, 1) dairies, wineries, breweries, and beverage plants, 2) meatand poultry processing and packing plants, 3) milk and dairy productsprocessing and packaging plants, 4) seafood and produce processing andpackaging plants, 5) food processing and packing plants, 6) eggprocessing and packing equipment surfaces, and 7) eating establishments.Microbial populations are reduced in continuous online applications orin discontinuous batch applications. The composition may be recoveredfor reprocessing/reapplication to poultry, meat, seafood, plantmaterials, and to surfaces.

Direct Contact with Food: The compositions are useful for treating andintervening with microbial vectors as applied to any kind of poultry(i.e., chicken, turkey, duck, ostrich, emu, quail, partridge, squab,guinea fowl, pheasant, goose, and game hens), red meat (i.e., beef,buffalo, pork, veal, lam, and mutton), and seafood (fish and shellfish).Also, to animal, poultry, or seafood carcasses that have been subjectedto stunning, bleeding, scalding, picking, singeing, or a combinationthereof Also, to meat and poultry that have been subjected to beheading,removing feet, neck-cropping, portioning, or a combination thereof.Also, to a whole carcass or individual parts of carcasses (parts) or oneor more dismembered parts of a carcass. Also, to a carcasses (parts)that has/have been subjected to portioning, or to any portion orcombination thereof that has been subjected to deboning.

The composition may also be applied to plant material (i.e., leaf,stalk, flower, fruit, vegetable, or portion thereof) for treating andintervening with the growth of microbial vectors.

Treatment of food material may be by any suitable means including, forexample, dip, spray nozzle or electrostatic spray system. Additionally,in poultry processing it may be applied within a de-feathering picker,inside-outside bird washing, dress rinsing, spray rinsing, or acombination thereof. Application may further include treating foodmaterial with the composition and then exposing the treated foodmaterial to activated light. Activated light is defined as ultravioletlight, infrared light, visible light or a combination thereof. Treatmentmay alternatively be by air chilling. The composition may be applied attemperatures between about 0° C. and 100° C. for use of the compositionwith direct food contact.

Surface applications: Surfaces prone to contamination withmicroorganisms can be found in many industries. Automated,clean-in-place (CIP) systems are widely used in food and beverage plantsthroughout product reception, storage, and process areas. This practicehas expanded the need for cold terminal sanitation of stainless steelstorage silos and process tanks, particularly in dairies and breweries.The advantages of CIP systems to the processors are savings inpersonnel, energy, and chemical costs by prevention of misuse byemployees. Potential difficulties encountered using CIP systems areeffective removal of soils, uniform surface contact by cleaners andsanitizers, problems with foam, and cross contamination problemsassociated with the practice of processing multiple product lines.

In these applications, the composition is applied to solid surfaces forthe control of microbiological organisms. Surfaces may be porous ornon-porous. One method for applying the composition is by a dual feedpump dosing system using ratio control or other means to proportionatelymeter the active formulations into a common stream of water or otherappropriate carrier for the peracetic acid and additives (salicylic andcitric) to be applied onto surfaces such as, but not limited to, any andall food processing equipment, bottle washing systems (including but notlimited to such systems for glass, HDPE, MDPE, LLDPE and PET, and PLA),food contact surfaces, or other non-food processing surfaces.Application to surfaces may be accomplished, with or without foamadditive, as treatments for food contact surfaces in restaurants,hospitals, and other industrial and institutional areas requiringsanitization and disinfection. Additionally, application may beaccomplished, with or without foaming additives, as treatments fornon-food contact surfaces such as, but not limited to, locker rooms,shower facilities, toilets, rest rooms, and counter tops.

The invention is exemplified in the following examples. The examples areillustrative of the invention and not intended to be limiting.

EXAMPLES

Methods and Materials:

“RB” refers to a mixture of 0.2% sodium salicylate (Mallinckrodt powder)and 0.5% citric acid (Sigma anhydrous crystal), each ingredient addedseparately as a solid.

P-077-22 is a solution comprised of 81% acetic acid, 8% water, 7.6%citric acid, and 3.0% salicylic acid. P-077-23 is a solution comprisedof 79% acetic acid, 14.2% water, 5.6% citric acid, and 1.2% sodiumsalicylate. In all studies, the “P” solutions were added such that thefinal test solutions contained 0.5% citric acid. For P-077-22 the actualweight of “as is” solution used was 6.58%; and for P-077-23 the weightwas 8.93%.

The acronym “POESM” refers to Tween 80 (polyoxyethylene 20 sorbitanmonooleate). The acronym “DDBSA” refers to dodecylbenzenesulfonic acid,and “SLS” refers to sodium lauryl sulfate.

Use of oxidative lysing agents such as peracetic acid in aqueoussolution at concentration(s) ranging from 400-660 ppm provided onlymoderate kill rates with log reductions of 4-5.5. However, use ofperacetic acid at the same concentrations with the addition of theadjuvant herein described produced improved kill rates up to completedestruction of the species with log reductions of 6 or recovery rates of0. Additional testing of peracetic acid at nominal rates of 0-200 ppmwith and without the adjuvant clearly demonstrated that complete controlagainst C. albicans was achieved only when both PAA and the adjuvantwere deployed together.

Additional testing to prove control as a disinfectant and not haveinterference between the adjuvant with the peracetic acid (VigorOx®LS&D, FMC, USA) was demonstrated through a 3^(rd) party laboratory withcomplete control of the tested organisms and met U.S. EPA disinfectionclaims. Similar testing also demonstrated similar performance with athird party laboratory as a sanitizer for pre-cleaned, non-porous foodcontact surfaces meeting the U.S. EPA definition for sanitizerperformance.

The method of deployment consisted of peracetic acid in a water carrierfluid to which has been added the adjuvant herein described and thenapplied to the surface(s) to be treated and held for a specific periodof time determined by field application testing. All the components werecompounded into a single homogeneous mixture.

In the examples, the combination of 0.2% sodium salicylate and 0.5%citric acid added as solids (RB) or as dissolved in acetic acid(P-077-23) significantly enhanced the efficacy of VigorOx® versusmicrobes with thick cell walls such as Gram-positive bacteria, yeasts,and molds. This increase in biocidal effectiveness is most likely due tocell wall damage/changes caused by the adjuvants, allowing furtherpenetration by peracetic acid. Since VigorOx® is highly/totallyeffective against thinner-walled Gram-negative bacteria, the addition ofadjuvants results in no/little difference in efficacy against thesemicrobes.

In parallel tests, the P-077-22 formulation containing salicylic acidachieved practically identical reductions as the sodiumsalicylate-containing version against all microbes except the spoilagemold Rhizopus oryzae. Thus, studies conducted to date indicate thatP-077-23 is the more effective formulation; however, it should be notedthat only one study versus Rhizopus indicated that P-077-22 wasinferior. (See Example 4.)

Disinfecting Test Method:

Laboratory Procedures

Preparation of challenge inoculi for IsoGrid and suspension efficacytesting

For bacterial studies, test microbes obtained from ATCC or Remel werefirst streaked on nutrient agar plates to confirm purity. Some of theplate growth was then transferred to nutrient broth and stirred at ˜37°C. for ˜24 hrs. or until growth reaches a level of 8 log₁₀ colonyforming units (CFU)/ml. This broth was then serially diluted in nutrientbroth with 5% calf serum (added as an organic burden) to prepare a ˜6log₁₀ CFU/ml suspension for the inoculation of test filters and a ˜2log₁₀ CFU/ml suspension for the inoculation of positive control filters.

For yeast studies, test microbes obtained from ATCC or Remel werestreaked on appropriate agar to confirm purity and then spread over theentire surface of several agar plates. After an incubation period ofsufficient length to yield heavy growth, the surface of the agar wasgently scraped with an inoculating loop to transfer the growth to a tubeof sterile Butterfield's buffer (generally one plate of growth in ˜100ml buffer). The concentration (CFU/ml) of the resulting suspension wasdetermined by using a hemacytometer or inoculating agar plates orIsoGrid filters. The mixture was refrigerated and was usable for severalweeks after preparation. On the day of testing, the suspension wasserially diluted in Butterfield's phosphate buffer to a level of ˜5-6log₁₀ CFU/ml for the inoculation of test filters and a ˜2 log₁₀ CFU/mlsuspension for the inoculation of positive control filters.

For mold studies, test microbes obtained from ATCC or Remel werestreaked on appropriate agar to confirm purity and then spread over theentire surface of several agar plates. After an incubation period ofsufficient length to yield confluent growth and sporulation, the plateswere flooded with 6-10 ml sterile Butterfield's buffer. A hockey stickplate spreader was used to gently scrape the surface of the agar toloosen conidia and hyphae. The suspension on the agar was then pouredoff and filtered through sterile gauze in a sterile Buchner funnel withreceiving flask. The concentration (CFU/ml) of the resulting conidialfiltrate was determined by using a hemacytometer or inoculating agarplates or IsoGrid filters. The mixture was refrigerated and was usablefor several weeks after preparation. On the day of testing, thesuspension was serially diluted in Butterfield's phosphate buffer to alevel of ˜5-6 log₁₀ CFU/ml for the inoculation of test filters and a ˜2log₁₀ CFU/ml suspension for the inoculation of positive control filters.

Preparation of Test Solutions

Peracetic acid (PAA) solutions were prepared by diluting 5% or 15%VigorOx® with “Milli-Q” water in a passivated amber bottle to obtain thetarget concentration. The concentration was then confirmed by titrationon an autotitrator.

Mixtures of PAA and amounts of adjuvants </=1 g/100 g mixture wereprepared by weighing the adjuvant directly into a passivated amberbottle and adding previously prepared peracetic acid to reach thedesired total weight. The PAA content was determined by titrating with atest PAA kit #7191 (LaMotte Company, Chestertown, Md. 21620 USA).

Mixtures of PAA and amounts of adjuvants >1=1 g/100 g mixture wereprepared by weighing the adjuvant and the peracetic acid separately intoa passivated amber bottle and using Milli-Q water to reach the desiredtotal weight. The PAA content was determined by titrating with a testPAA kit #7191 (LaMotte Company, Chestertown, Md. 21620 USA).

IsoGrid Efficacy Testing

Using aseptic technique, IsoGrid filters (enough to run each testsolution/condition in triplicate), were clamped in filter funnel unitsand each inoculated with a tartet of 100,000 to one million CFU. Thiswas accomplished by first adding ˜12 ml sterile Butterfield's buffer tothe funnel followed by 1 ml of ˜10⁵-10⁶ CFU/ml inoculum suspension, andthen removing all fluid under vacuum. Positive controls were similarlyprepared by adding 1 ml of a ˜10² CFU/ml suspension. Afterwards, filtersare transferred to empty sterile Petri dishes and dried with dish lidscracked open for ˜15 min. in a biosafety cabinet. For treatment, eachdried test filter is mounted in a filter funnel unit to which 15 ml oftest biocide solution is then added. After the desired treatment time,the biocide is neutralized by the addition of 50 ml of Letheen brothwith 0.5% sodium thiosulfate; all fluid removed under vacuum, and thefilter transferred to an agar plate. The plates are enumerated after anappropriate incubation period.

Suspension Efficacy Testing

For this type of study, one ml of challenge inoculum (-6-7 log₁₀ CFU/ml)is mixed with nine ml of biocide in a sterile tube at Time 0. Atprecisely timed intervals, one ml aliquots of this reaction mixture aretransferred to sterile tubes containing 9 ml neutralizing solution(Letheen broth with 0.5% sodium thiosulfate). Afterwards, the contentsof each neutralizer tube are poured onto an IsoGrid filter mounted in afilter funnel unit (as used in the IsoGrid testing described above).Each tube is twice rinsed with sterile Butterfield's buffer and therinsate added to the funnel unit before all fluid is removed undervacuum. Positive controls are prepared by serially diluting the ˜6-7log₁₀ CFU/ml inoculum suspension in Butterfield's buffer to make a ˜2log₁₀ CFU/ml suspension. One ml of this suspension is then used toinoculate triplicate IsoGrid filters. All filters arc transferred toagar plates, incubated, and enumerated.

The following tables list the reductions achieved by test biocidesagainst several types of microbes in both IsoGrid and suspensionstudies.

Example 1

VigorOx® at low concentrations with adjuvant showed significant enhancedactivity against these spoilage bacterial organisms. All tests were runin triplicate. Tests were conducted at room temperature and for 30 sec.

TABLE 1.1 Species Avg. LOG₁₀ Avg. LOG₁₀ Biocide Treatment ChallengeRecovery Reduction [—] Control (FMC table 1) E. coli 6.4206 — 85 ppm PAAVigorOx ® (5%) E. coli 0.0001 −6.4205 85 ppm PAA VigorOx ® (5%) + RB E.coli 0 −6.4206 Control P. aeruginosa 7.5714 — 85 ppm PAA VigorOx ® (5%)P. aeruginosa 0.9736 −6.5978 85 ppm PAA VigorOx ® (5%) + Adjuvant P.aeruginosa 0 −7.5714 (RB, P-077-22 and P-077-23 all got complete kills)Table 4 FMC data Control (FMC Table 5) S. aureus* 5.9403 — 85 ppm PAAVigorOx ® (5%) S. aureus* 4.5858 −1.3544 85 ppm PAA VigorOx ® (5%) +P-077-23 S. aureus* 0.2596 −5.6807 *S. aureus past expiration date.

Example 2

Candida parapsilosis vs. VigorOx® and VigorOx® with Adjuvant.

The VigorOx® with adjuvant showed significant enhanced activity againstthis spoilage yeast organism. Testing at room temperature showedcomplete kill at the three time intervals and no residual for futurecolonization.

TABLE 2.1 Candida Count Count Count parapsilosis (MPN) (MPN) (MPN)Biocide Challenge 30 sec. 45 sec. 60 sec. 440 ppm PAA 15% 1.69 × 107TNTC TNTC TNTC VigorOx ® 440 ppm PAA VigorOx ® 1.69 × 107 0 0 0 withAdjuvant

Example 3

Modified Suspension Efficacy Study vs. Mucor plumbeus

Mucor plumbeus is a problematic spoilage yeast. 1 ml 1.1×10⁷ cfu/mlMucor plumbeus suspension was added to 9 ml of prepared biocide at time0. 1 ml of reaction mixture was transferred to a tube containing 9 mlLetheen plus 0.5% sodium thiosulfate at 30 sec., 45 sec., and 60 sec.Each tube of neutralized mixture was used to inoculate an IsoGridfilter. Tube was then rinsed with Butterfield's buffer twice and therinsate poured back into the IsoGrid funnel and removed under vacuum.

Filters were plated on malt agar and incubated at room temperature for 5days. Inoculum suspension was serially diluted in Butterfield's bufferand the 10⁻⁶ dilution plated on triplicate IsoGrid filters.

TABLE 3.1 Modified Suspension Efficacy Study vs. Mucor plumbeus CountCount Count Mucor (MPN) (MPN) (MPN) Biocide Challenge 30 sec. 45 sec. 60sec. 440 PAA 5% VigorOx ® 1.1 × 10⁷ TNTC TNTC TNTC 440 PAA 5%VigorOx ® + 1.1 × 10⁷ 0 0 0 Adjunct test A 440 PAA 5% VigorOx ® + 1.1 ×10⁷ 0 0 0 Adjunct test B TNTC = too numerous to count

Example 4

R. oryzae Challenge with VigorOx® & VigorOx® with Adjuvant 1 ml 2.69×10⁷cfu/ml Rhizopus oryzae mold suspension was added to 9 ml of preparedbiocide at time 0. 1 ml of reaction mixture was transferred to a tubecontaining 9 ml Letheen plus 0.5% sodium thiosulfate at 30 sec., 45sec., and 60 sec. Each tube of neutralized mixture was used to inoculatean IsoGrid filter. The tube was then rinsed with Butterfield's buffertwice and the rinsate poured back into the IsoGrid funnel and removedunder vacuum. Filters were plated on malt agar and incubated at roomtemperature for 1 day. Inoculum suspension was serially diluted inButterfield's buffer and the 10-5 dilution plated on triplicate IsoGridfilters.

TABLE 4.1 R. oryzae Challenge with VigorOx ® & VigorOx ® with AdjuvantCount Count Count Rhizopus (MPN) (MPN) (MPN) Biocide Challenge 30 sec.45 sec. 60 sec. 440 ppm PAA 5% 2.69 × 10⁷ TNTC TNTC TNTC VigorOx ® 440ppm PAA VigorOx ® 2.69 × 10⁷ 0 0 0 with Adjuvant TNTC = too numerous tocount

What is claimed is:
 1. A composition comprising, in aqueous solution, a.an aqueous peracetic acid solution; and b. an adjuvant comprising (i)salicylic acid or a salt thereof and (ii) citric acid or a salt thereof.2. The composition of claim 1 wherein the salicylic acid and citric acidor their respective salts are dissolved to their solubility limits andin proportionate weight ratios ranging from about 1:5 or 1:1respectively.
 3. The composition of claim 2 further comprising one ormore stabilizing agents, wetting agents, hydrotopes, thickeners, foamingagents, acidifiers, pigments, dyes, surfactants, or combinationsthereof.
 4. The composition of claim 3 wherein the surfactant consistsof one or more anionic or nonionic surfactants in concentrations up to2% selected from the group consisting of polyoxylene (C₂₀ to C₈₀)sorbitan monooleates, alklyl long chain fatty acid (C₅ to C₂₀) metallicesters and alcohols; and alkylbenzenesulfonic acids and alcohols and/ortheir metallic ester salts having alkyl groups ranging from C₂ to C₂₀.5. The composition of claim 1 wherein the salicylate salt or salicylicacid and citric acid compounds are in proportionate weight ratiosranging from 1:5 to 1:1 respectively, in a solution matrix of glacialacetic acid (70-90%) and water (30-5%).
 6. The composition of claim 1wherein the salicylic acid or salt thereof is sodium salicylate and thecitric acid or salt thereof is citric acid.
 7. A kit comprising a. anaqueous peracetic acid solution; and b. an adjuvant comprising (i)salicylic acid or a salt thereof and (ii) citric acid or a salt thereof.8. A method of reducing microbial contamination comprising applying thecomposition of claim 1 to a surface in an amount and for a timesufficient to reduce the microbial contamination.
 9. The method of claim8 wherein the surface is meat, poultry, seafood, or a portion thereof.10. The method of claim 8 wherein the surface is a plant material (leaf,stalk, flower, fruit, vegetable, or portion thereof).
 11. The method ofclaim 8 wherein the surface is a non-food contact surface.
 12. Themethod of claim 8 further comprising recovering the applied compositionand reprocessing the recovered composition to yield a recycledanti-microbial composition.
 13. The method of claim 8 wherein theperacetic acid is present at end use concentrations ranging to 1%. 14.The method of claim 8 wherein the salicylic acid or salt thereof ispresent at up to 20000 ppm.
 15. The method of claim 8 wherein the citricacid is present up to 20000 ppm.
 16. The method of claim 8 wherein theend use concentrations of the composition have pH values between 1 and4.5.